It could be said that hydrogels that can be prepared under mild conditions for organisms and cells are very attractive materials in the medical field. Methods that utilize an enzyme reaction are attracting considerable attention as techniques for obtaining these types of hydrogels. Among such methods, the development of materials that use an enzyme reaction of horseradish peroxidase (HRP) derived from horseradish in crosslinking reactions between polymers has been widely reported.
HRP is an enzyme that uses hydrogen peroxide as a substrate to catalyze oxidative coupling reactions between phenol, aniline and thiol. The hydrogen peroxide that is generally required for the reaction to proceed is typically supplied by adding a hydrogen peroxide solution. However, in this type of method, it has been reported that because the hydrogen peroxide concentration temporarily reaches a high level within the system, the HRP may become deactivated, resulting in the crosslinking reaction not proceeding satisfactorily (Non-Patent Document 1). Moreover, it is also thought that when a cell or protein is enveloped within the hydrogel, a high concentration of hydrogen peroxide may have an effect on the enveloped structure.
Accordingly, in recent years, new methods for preparing hydrogels have been reported in which the HRP catalytic cycle is proceeded without adding hydrogen peroxide (Non-Patent Documents 2 and 3). In Non-Patent Document 2, a glucose oxidase (GOx) is used, and the hydrogen peroxide generated when the GOx oxidizes glucose is used to proceed the HRP catalytic cycle, thereby succeeding in gelling an aqueous solution of a polymer having introduced phenolic hydroxyl groups. In this method, because the hydrogen peroxide is generated gradually within the system, deactivation of the HRP can be suppressed to a minimum. Further, it is also reported that because the crosslinking density increases in the produced hydrogel, the hydrogel has superior mechanical properties compared with hydrogels produced by conventional methods in which hydrogen peroxide is added.
In Non-Patent Document 3, it is reported that a hydrogel can be produced by the extremely simple method of merely mixing a thiol group (SH)-modified polymer and HRP. This method initially uses the hydrogen peroxide generated by SH self-oxidation to proceed the HRP catalytic cycle and produce a hydrogel. Further, it is thought that because hydrogen peroxide is also produced during the HRP catalytic cycle, there is no need to add hydrogen peroxide to the system, thus providing an extremely simple gel production method. However, in the method of Non-Patent Document 3, a hydrogel cannot be produced under physiological conditions of pH 7.4, and a hydrogel cannot be produced unless the pH is 8.5. Furthermore, other problems arise in that a high concentration of HRP (>1.4×103 U/mL) and a long gelation time (>2 h) are required.
It is thought that the reason for these problems is that the HRP catalytic cycle does not proceed efficiently. FIG. 1 is a diagram from Non-Patent Document 4, and illustrates the catalytic cycle when HRP oxidizes SH. In FIG. 1, the rate constant of reaction 3 (HRP (COMP II)+RSH→HRP (Fe3+)+RS) is extremely low at 300 M−1 s−1, and it is thought that this is the cause of the problems mentioned above.
In Non-Patent Document 4, it is reported that adding homovanillic acid during the HRP SH catalytic cycle accelerates the oxidation of SH. In Non-Patent Document 4, it is suggested that this result is due to an acceleration in the production of thiol radicals by a radical rearrangement reaction from phenoxy radicals produced in the system to thiol radicals (Ph-O+RSH→Ph-OH+RS).
Moreover, in Non-Patent Document 5, it is reported that in a system using tyrosine as a phenol derivative and glutathione (GSH) as a thiol derivative, the rate constant for the radical rearrangement reaction from a phenoxy radical to a thiol radical is approximately 2×106 M−1 s−1. Further, the rate constant when the activated HRP (HRP (COMP (II)) recognizes phenol (HRP (COMP II)+Ph-OH→HRP (Fe3+)+Ph-O) is approximately 104 to 106 M−1 s−1, indicating that the rate constants in these two reactions are much larger than the rate constant for the direct oxidation reaction of SH by HRP (COMP (II)) (reaction 3 in FIG. 1).
On the other hand, Patent Document 1 discloses a polysaccharide hydrogel that is a condensation polymer of a polysaccharide such as hyaluronic acid and a polymerizable compound, and also discloses a method for producing the polysaccharide hydrogel by binding a polymerizable compound to the polysaccharide using a condensing agent, and then treating the resulting mixture with oxygen to polymerize and cure the compound.